Assay methods for detection of a virus in an avian tissue sample

ABSTRACT

The invention relates to methods of detecting a virus in an avian tissue sample wherein genetic material derived from feathers is tested for the presence of genetic material from the virus.

The present invention relates to assay methods and, in particular, tomethods for detecting the presence of a virus, especially Marek'sdisease virus (MDV), in an avian tissue sample.

BACKGROUND

Marek's disease virus (MDV) is a herpesvirus, which causeslymphoproliferative disease in chickens. Even after the introduction ofvaccines against MDV, the infection still causes considerable losses inthe poultry industry. MDV is divided into three serotypes, all of whichestablish latent infections. Serotype 1 includes oncogenic viruses,serotype 2 non-oncogenic viruses and serotype 3 includes the turkeyherpesviruses (HVT) (Bülow et al (1976) Zentralblatt fürVeterinarmedizin, 23B, 391-402).

The traditional diagnosis of Marek's disease is based on the clinicalsigns and pathological alterations. However, more specific methods forsurveillance of the prevalence of MDV would be desirable. The detectionof viral antigen in the feather follicle epithelium by the agar gelprecipitation test has been described by Haider et al (1970) PoultryScience, 49, 1654-1657. The different serotypes can be differentiated bythe agar gel precipitation test (Lee et al (1983) Journal of Immunology,130, 1003-1006), but the sensitivity of that test is inferior to that ofenzyme-linked immunosorbent assay (ELISA) and DNA hybridization(Davidson et al (1986) In: Current research on Marek's disease.Proceedings of the 5^(th) International Symposium on Marek's Disease(pp. 311-316). Tallahassee: Rose Printing Company, Inc.).

The preferred samples for virus isolation are buffy-coat cells, whichcan be co-cultivated with susceptible primary cell cultures.Immunofluorescent assay (Kitamoto et al (1979). Biken Journal, 4,137-142) or ELISA (Cheng et al (1984) Avian Diseases, 4, 900-911), canbe used for subsequent identification of the MDV serotype.Alternatively, the serotype can be identified by restrictionendonuclease analysis (Ross et al (1983). Journal of General Virology,64, 2785-2790) or polymerase chain reaction (PCR) (Wang et al (1993)Molecular and Cellular Probes, 7, 127-131. In situ hybridization hasbeen used for detection of MDV genome in infected tissue (Endoh et al(1996) Journal of Veterinary Medical Science, 58, 969-976; Ross et al(1997) Journal of General Virology, 78, 2191-2198), but this techniqueis probably too laborious for routine diagnoses. (Davidson et al (1995)Avian Pathology, 24, 69-94; and Davidson et al (1996) In: Currentresearch on Marek's disease. Proceedings of the 5^(th) InternationalSymposium on Marek's Disease (pp. 311-316). Tallahassee: Rose PrintingCompany, Inc.), applied MDV serotype 1-specific PCR techniques to fullblood and tumour tissue samples from commercial chicken and turkeyflocks, the majority of which had neoplastic disease. Wang et al (1993)Molecular and Cellular Probes, 7, 127-131; Young, P. & Gravel, J. (1996)In Current research on Marek's disease. Proceedings of the 5^(th)International Symposium on Marek's Disease (pp. 308-310). Tallahassee:Rose Printing Company, Inc.; and Silva, R. F. & Witter R. L. (1996) InCurrent research on Marek's disease. Proceedings of the 5^(th)International Symposium on Marek's Disease (pp. 302-307). Tallahassee:Rose Printing Company, Inc., applied a MDV serotype 1-specific PCRprotocol to various tissues of chickens experimentally inoculated withthe JM/102 strain.

Handberg et al (2001) Avian Pathology 30: 243-249 describe the use ofserotype 1- and serotype 3-specific PCR for the detection of MDV inchickens. Tissue samples were taken from blood (buffy-coat cells),spleen, liver, skin, feather tips and ovaries.

DESCRIPTION OF THE INVENTION

The present invention provides further methods for detecting a virus,especially MDV, in avian tissue samples.

In a first aspect the invention provides a method of detecting a virusin an avian tissue sample comprising: extracting genetic material froman avian tissue sample; and testing the extracted genetic material todetect any genetic material from the virus; characterised in that theavian tissue sample is derived from one or more feathers of the axillarytract.

By using feather samples the test can be carried out on live animals.Sampling of feathers is simple, quick and practical under fieldconditions. Feather samples can be placed in a suitable container andtested immediately or stored for future testing, as desired. Bycontrast, sampling blood requires great care to prevent blood clotsforming, including transport of blood under cool, controlled conditions.Blood clotting leads to negative test results. Internal organ samples,such as spleen and tumour samples, must be transported on wet ice, whichis impractical under field conditions.

By selecting axillary tract feathers from which to derive a tissuesample, the invention provides significant advantages over known methodswhich take tissue samples from different parts of the bird.

Surprisingly, we have found that virus can be detected in axillary tractfeathers at higher levels than in other feathers and therefore virus canbe detected in axillary tract feathers according to the invention whenit cannot be detected in other tissue samples, including other feathers.Accordingly, the methods of the invention are particularly suitable formonitoring the extent to which a flock of birds has been immunisedeffectively with MDV vaccine, by detecting the presence of the vaccinestrain in axillary tract feather tissue samples.

By “avian” we include any bird, but preferably birds which are producedcommercially, especially poultry such as chickens, turkeys, ducks, etc.

By “axillary tract feathers”, we include the meaning of the featherslocated in the region of a bird marked “axillary” in the accompanyingfeathering diagram (FIG. 1). Preferably, the axillary tract featherselected is a “pin feather”, that is, an immature growing feather. Theterm “pin feather” will be familiar to skilled persons. For example, vanTyne J & Berger A J (1959) Fundamentals of Ornithology, John Wiley, NewYork refer to a pin feather as “a new, growing feather, still notcompletely unsheathed”. Lucas A M & Stettenheim P R (1972) AvianAnatomy, Integument Part 1, US Government Printing Office, Washington,pp 199-200 remark “The new feather is tightly furled inside a sheathwhile it forms. As it appears above the skin, it has a long conicalshape with a blunt tip and a slightly moist surface. A feather at thisstage in any generation is often called a pin feather”. (See also FIG.1( a), which is taken from Lucas & Stettenheim (1972)).

Preferably, the axillary tract feathers are taken from chicks which areadvantageously less than a month old.

In a preferred embodiment, the method is performed on samples taken fromchicks on or prior to 13 days post-immunisation, preferably at between 8days to 12 days post-immunisation, more preferably at between 9 to 11days post-immunisation, i.e. on days 9, 10 or 11 post-immunisation.

Preferably, the method provides quantitative information on the amountof virus, especially MDV, in the sample.

Preferably, the method is specific for MDV serotype 1, and morepreferably the method is specific for MDV-1 Rispens strain CVI 988. Thelatter strain is a commercial vaccine strain produced by Fort Dodge,Iowa, USA which is available from the American Type Culture Collection(ATCC), Mannassas, Va., USA.

Advantageously, the method involves the use of a PCR reaction.Preferably, before said PCR reaction is carried out, the extractedgenetic material to be tested is treated with an agent to overcome theinhibitory effect of any feather tissue factor which may be present.This inhibitory effect appears to be associated with melanin and cantherefore be a particular problem when feathers from brown birds aresampled. Preferably, the agent is selected from one or more of bovineserum albumin; porcine (pig) albumin; and ovine (sheep) albumin.

Skilled persons will be aware of a range of detection methods fordetecting viral, especially MDV, genetic material which could be used inthe methods of the invention, such as the methods of Handberg et al(2001) supra. A particularly preferred method for detecting MDV-1 strainCVI 988 is as follows:

-   -   (i) providing forward and reverse primers for a nucleic acid        polymerase, which primers are selected from the nucleotide        sequence which flanks the 132 bp repeat nucleotide sequence of        MDV;    -   (ii) amplifying nucleic acid sequences between the primers;    -   (iii) detecting the number of 132 bp repeat sequences in the        amplified nucleic acid sequences; and    -   (iv) relating the number of 132 bp repeat sequences to the        identity of the viral nucleic acid and thereby identifying the        type of MDV in the tissue sample, multiple copies of the 132 bp        repeat sequence being indicative of MDV-1 strain CVI 988.

A preferred quantitative method for use in detecting MDV according tothe present invention comprises:

-   -   (a) providing a polynucleotide sequence which is capable of        binding specifically to a MDV-specific target polynucleotide;    -   (b) contacting the extracted genetic material with a probe        whereby the probe binds specifically to its target MDV        polynucleotide;    -   (c) determining whether the probe has bound to its target MDV        polynucleotide; and    -   (d) determining whether the sample contains MDV on the basis        that the presence of the target polynucleotide indicates the        presence of MDV in the sample.        Step (d) preferably provides a quantitative determination of the        amount of virus in the sample.

Advantageously the step of determining whether the probe has bound to atarget polynucleotide comprises amplifying a region of the targetpolynucleotide, which region comprises the binding site of the probe.

Preferably the probe has the sequence 5′ AGA CCC TGA TGA TCC GCA TTG CGACT 3′ (SEQ ID No. 1).

Preferably, amplification is primed by the following primers:

-   -   Forward primer (GGT CTG GTG GTT TCC AGG TGA—SEQ ID No. 2) which        is located at NT positions 1341-1361 in the GA strain Meq gene        sequence.    -   The GA (Georgia) strain was a 1964 isolate from Georgia, from an        ovarian tumour. Reference: C. S. Eidson & S. C. Schmittle        (1968). Studies on acute Marek's disease. I. Characteristics of        isolate GA in chickens. Avian Diseases 12; 467-476.    -   Reverse primer (GCA TAG ACG ATG TGC TGC TGA—SEQ ID No. 3) is        located at NT positions 1413-1393.

Advantageously, the probe is labelled fluorescently and the step ofdetermining whether the probe has bound to a target polynucleotidecomprises determining the fluorescent emissions of the probe.

Two fluorescent dyes are brought into physical proximity by directconjugation at opposite ends of a short oligo probe (5′ reporterfluorochrome, usually 6-FAM, and 3′ quencher fluorochrome, usuallyTAMRA). When the high-energy fluorophore (FAM) is excited at 488 nm,instead of the expected fluorescence emission at 520 nm the capturedenergy is transferred to the lower energy fluorophore (TAMRA) and isemitted at 580 nm (fluorescence resonance energy transfer, FRET, hasoccurred). Using the fluorescein/rhodamine reporter/quenchercombination, FRET will effectively occur even when the groups areseparated by 25-30 bases of DNA. During the course of a TaqMan™ assaythe two fluorophores are physically detached from each other by the5′-nuclease action of Taq DNA polymerase—after which 488 nm stimulationresults in visible FAM emission at 520 nm.

Dual-labeled probes usually have a 5′-reporter dye, such as FAM, TET,HEX, JOE or VIC and a 3′-quencher group, such as TAMRA or Dabcyl (auniversal quencher).

Suitable fluorescent labels are within the common general knowledge ofskilled persons. The following reagents are available fromSigma-Aldrich, UK: HEX stands for hexachloro-fluorescein; TET stands fortetrachloro-fluorescein; Joe is6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein; ROX is5-carboxy-Rhodamine; dabcyl is 4-((4-(dimethylamino)phenyl)azo)benzoicacid.

Structures and further information on all of these are available on themolecular probe website www.probes.com. VIC dye is available fromApplied Biosystems.

6-FAM=6-carboxyfluorescein; (a phosphoramidite); yellow-green dye;absorbance maximum=494 μm, emission maximum=525 nm

HEX=a phosphoramidite; pink dye; absorbance maximum=535, emissionmaximum=556

TET=a phosphoramidite; orange dye; absorbance maximum=521, emissionmaximum=536

VIC=absorbance maximum 538, emission maximum=554

JOE=absorbance maximum 521, emission maximum=547

TAMRA=6-carboxy-tetramethyl-rhodamine; absorbance maximum=555, emissionmaximum=580

Dabcyl=absorbance maximum=453, no maximum emission (universal quencher)

From the foregoing description it will be apparent that an importantaspect of the methods of the invention is the use of avian tissuesamples which are more convenient and useful than avian tissue samplesused previously for detecting viral, especially MDV, infection.Accordingly, further aspects of the invention relate to the provision ofavian tissue samples.

In a second aspect the invention provides an isolated avian tissuesample from one or more feathers from the axillary tract.

By “isolated” we include the meaning that the tissue sample is free of asubstantial amount of the material with which it is normally associatedin nature. For example, the tissue sample may be stored in a container,or be derived from the original axillary tract feather by a variety ofisolation; extraction and/or purification methods.

Preferably the isolated tissue sample consists of the proximal portion(the non-barbed portion which is attached to the skin and which containsthe pulp—see accompanying figures). Accordingly it is preferred that theproximal portion of the axillary tract feather is isolated from thedistal (barbed) portion of the feather. This is easily achieved simplyby cutting off the proximal portion of the feather with a pair ofscissors and discarding the distal portion.

In a third aspect the invention provides a genetic material-containingextract from an avian tissue sample wherein the extract is taken from asample of tissue as described in relation to the second aspect of theinvention.

It will be appreciated that samples according to the second and thirdaspects of the invention may be collected and/or prepared in the field,or transported to a separate location, such as a laboratory, forpreparation and testing. Hence, further aspects of the invention relateto samples according to the second and/or third aspects of the inventionstored in a form suitable for transport to a separate location.

The feathers could be stored complete (e.g. in 20 ml Sterilin universalplastic tubes), or after cutting off the proximal portion required forDNA preparation (e.g. in 1 ml Eppendorf snap-cap or screw-cap tubes).Alternatively, the feathers could be stored in heat-sealed or tiedplastic bags. For short-term storage (e.g. 1 week), the feathers couldbe stored at 4° C., but, for longer periods of storage, they should bestored frozen at −20° C.

The DNA should be stored at −20° C. in screw-cap or snap-cap 0.5 ml or1.5 ml Eppendorf tubes, in Tris-EDTA buffer or, if the DNA is to be usedin Taqman analysis, in water since EDTA will inhibit the Taqmanreaction.

Advantageously, the results of the methods are furnished in anintelligible format. Preferably, the results are recorded or stored onan information carrier. However, the step of furnishing the resultscould be by communicating the results orally.

By “information carrier”, we include any means of storing information,such as paper, a computer disk; an internet-based information transfersystem, such as an e-mail or internet page, or electronic file, etc. Ofcourse, an “intelligible format” is also intended to embrace encryptedinformation which can be deciphered with an approximate key.

Examples embodying certain aspects of the invention will now bedescribed with reference to the following figures in which:

FIG. 1( a) shows the development of feathers above the skin, during thefirst four generations. A circle indicates a feather that is stillgrowing; a dot, a feather that is fully grown.

FIG. 1( b) is a diagram of the feathering pattern in chickens whichshows the axillary tract.

The axillary tracts lie on the underside of the chick and extend, eachside, from the lower neck to the upper abdomen, underneath the wings andalongside the breastbone. FIG. 1 is taken from the book ‘Bird Structure:An approach through evolution development and function in the fowl’. D.A. Ede, Publisher: Hutchinson Educational, 1964. The feathering patternis the same in all birds.

FIGS. 2( a) to 2(d) are photographs of the axillary tract feathers andclose ups of individual feathers showing the portions which are retainedto provide tissue samples according to the invention. FIGS. 2( a) and2(b) demonstrate the axillary tract feathers. FIGS. 2( c) and 2(d)demonstrate the part of the feather taken for analysis.

FIG. 3:

Shows a dose response of serotype CVI988 vaccine as detected by Meqprimers (78 bp product) with Taqman technique. CVI988 vaccine washarvested from a bacterial artificial chromosome (BAC) vector.

This experiment was performed to test/optimise the use of the Meq Taqmanprimer/probe set. BAC10 DNA was used as the target DNA since it wasknown to contain many copies of the viral genome.

Taqman assay was used to detect the Meq gene in DNA derived from Rispensvirus genome cloned into a Bacterial Artificial Chromosome (BAC10). TheDNA was used in ten-fold dilutions. During each cycle of real-time PCR,the reporter fluorochrome FAM is released and able to fluoresce.Therefore, with each cycle, fluorescence intensity increases. The Ctvalue (the cycle at which fluorescence passes a fixed threshold) is ameasure of the starting copy number of the target sequence. The lowerthe Ct value, the higher the starting copy number of target sequence. ACt value of 40 indicates that no target sequence, or an undetectableamount of target sequence, is present. This figure shows that dilutionsof this DNA preparation between 1:10 and 1:10000 gave a detectableamount of Meq PCR product. The Ct value increases linearly withincreasingly dilute DNA.

FIG. 4:

Dose response of CVI988 vaccine in chicken embryo fibroblasts (CEF) andbacterial artificial chromosomes (BAC). This experiment was performed totest/optimise the use of the Meq Taqman primer/probe set on DNA derivedfrom Rispens-MDV infected cells.

Taqman assay was used to detect the Meq gene in DNA derived from BAC10,and in DNA derived from Rispens MDV-infected chick embryo fibroblast(CEF) cells. The DNA was used in ten-fold dilutions. This figure showsthat for both BAC10 DNA and Rispens-infected CEF DNA, the Ct valueincreases linearly with increasingly dilute DNA.

FIG. 5:

Dose response of CVI988 vaccine in chicken spleen and CEF. Thisexperiment was performed to test/optimise the use of the Meq Taqmanprimer/probe set on DNA derived from tissue samples from Rispens-MDVinfected chickens.

Taqman assay was used to detect the Meq gene in DNA from a spleen of aRispens-inoculated chick (11 dpi), and an age-matched uninoculatedchick. DNA from Rispens-infected CEF cells was used as a positivecontrol. The DNA was used in ten-fold dilutions of 1 mg/ml stocks and 1μl was used per reaction. Although the Ct values for the uninoculatedspleen DNA were lower than 40, they clearly did not increase withincreasing concentrations of DNA. However, Meq detection in theinoculated spleen DNA rose significantly above this baseline when theDNA was used neat, or at 1:10 dilution. We thus established that, forTaqman analysis of DNA taken from tissue samples of MDV-inoculatedchicks, we would use 1 μl DNA from a 1 mg/ml stock (i.e. 1 μg) DNA perreaction.

FIG. 6:

Time-course of replication of CVI988 vaccine in chicken feathersdemonstrating peak replication 15-20 days post infection. Thisexperiment was performed to follow the time-course of RispensMDV-infection in feather axillary tracts of inoculated chicks by Taqmanassay.

Taqman assay was used to detect the Meq gene in DNA prepared fromfeather tips of chicks at 0 (uninoculated), 10, 15, 20 and 28 dpi postinoculation with Rispens. A group of five chicks were sampled at eachtime-point (four chicks at 0 dpi). 1 μg DNA was used in the Taqmanassay. Mean Ct values for each group are plotted. The Ct values decreasefrom 0-15 dpi, then increase again from 20-28 dpi, showing a peak ofinfection 15-20 dpi.

FIG. 7:

This experiment was performed to follow the time-course of RispensMDV-infection in feather axillary tracts of inoculated chicks by 132 bprepeat PCR.

DNA was prepared from feather tips of Rispens-inoculated chicks at 0(uninoculated), 10, 15, 20 and 28 dpi. A group of five chicks weresampled at each time-point (four chicks at 0 dpi). 1 μg DNA was used inPCR (including 10 μg BSA per reaction) and the samples run on a 1%agarose gel containing ethidium bromide.

(M)=Lambda molecular size markers, (−)=water (negative control),(+)=Rispens BAC10 DNA (positive control). Days post infection areindicated underneath the gels.

(a) To confirm the PCR-quality and quantity of each DNA sample, PCR wasperformed to detect an endogenous retrovirus sequence present in allchicken cells. The 360 bp endogenous retrovirus product was detected inall of the feather samples confirming the PCR-quality of each samples.

(b) 132 bp repeat PCR was performed. The lane marked (+) shows the 132bp repeat ladder PCR products obtained with Rispens BAC10—six copies areclearly distinguished. The negative control shows no PCR product, as dothree of the four uninoculated chicks. The fourth uninoculated chickshows a faint product band equivalent to 3 copies of the repeat,indicating that the chick was contact-infected by the inoculated chickshoused in the same room. All of the inoculated chicks were 132 bp repeatpositive at 11, 15, 20 and 28 dpi. The PCR product representing acertain repeat copy number predominated in many cases and the number ofcopies represented by this predominant product varied between samples.This indicates that sub-clones of the inoculum virus, with a set numberof repeats, come to predominate in different chicks.

FIG. 8:

Graph showing a standard curve for Meq gene reaction using Rispens BAC10DNA

FIG. 9:

Graph showing a comparison of Rispens virus load in various feathertracts of individual chicks at (A) 8, (B) 13, (C) 19 and (D) 26 dayspost vaccination.

FIG. 10:

Graph showing Rispens virus load in various feather tracts determined byreal time PCR for Meq gene plotted as (A) on a logarithmic scale and (B)on a linear scale. Mean values (+SEM) for four vaccinated chicks.

EXAMPLE 1 Quantitative PCR Assay for MDV-1

Taqman™ quantitative PCR is an established technique used to quantifythe amount of starting PCR target by determining the number of PCRcycles required to reach a fluorescence threshold (definedmathematically by the Ct value). A higher copy number of target sequencein the sample requires fewer PCR cycles to reach the Ct threshold.

The Taqman primers and probe were designed from the Meq gene sequence ofthe MDV strain GA (see later). This sequence is published in: Jones D,Lee L, Liu J L, Kung H J, Tillotson J K. Marek disease virus encodes abasic-leucine zipper gene resembling the fos/jun oncogenes that ishighly expressed in lymphoblastoid tumors. Proc Natl Acad Sci USA. 1992May 1; 89(9):4042-6. The Sequence Accession No. is: M89471 (SEQ ID No.9). The Applied Biosystems ‘Primer Express’ software was used to selectthe optimum primer/probe sequences from the Meq sequence.

The specific primers used in this analysis multiply a 73 bp sequence inthe meq gene of MDV serotype 1 virus. This region is common to bothvaccine strains and field isolates.

The experiments described use CVI 988 vaccine as an example of serotype1 Marek's disease virus. The results can be applied to all serotype 1Marek's viruses because the area of the sequence the primers aredirected against is conserved in MDV serotype 1 viruses.

Protocol for Taqman™ Meq Gene PCR Analysis on Feather Tip Samples

1. Materials Required:

Reagents:

-   -   TNE buffer (store at room temperature) contains Tris (10 mM),        NaCl (150 mM), EDTA (1 mM) and the pH is adjusted to pH 7.5        using HCl    -   Sodium Dodecyl Sulphate (SDS) 10% solution (store at room        temperature)    -   Proteinase K (lyophilised powder from Sigma # P-6556, and make        up a stock of 20 mg/ml in water, stored at −20° C.)    -   Phenol pH 7.9 obtained from Sigma (catalogue no. P-4557) stored        at 4° C.)    -   Chloroform (stored at −20° C.)    -   3M Sodium Acetate pH5.2 (stored at room temperature)    -   Filtered neat ethanol (stored at room temperature)    -   Filtered 70% cold ethanol (stored at 4° C.)    -   PCR quality water    -   PCR quality water containing 800 μg/ml Bovine Serum Albumin        (BSA), filtered    -   Ice    -   Taqman™ PCR core reagents—Taqman™ buffer, MgCl₂, dNTPs, Taq        polymerase, Uracil N-Glycosylase (Perkin Elmer Biosystems)        -   Meq forward primer 5′ GGT CTG GTG GTT TCC AGG TGA 3′ (SEQ ID            No. 2) (MWG Biotech)        -   Meq reverse primer 5′ GCA TAG ACG ATG TGC TGC TGA 3′ (SEQ ID            No. 3) (MWG Biotech)        -   Meq probe 5′ FAM AGA CCC TGA TGA TCC GCA TTG CGA CT 3′ (SEQ            ID No. 1) TAMRA        -   (Sigma-Genosys Ltd)        -   (FAM & TAMRA are the fluorescent tags)

6-FAM=6-carboxyfluorescein; (a phosphoramidite); yellow-green dye;absorbance maximum=494 nm, emission maximum=525 nmTAMRA=6-carboxy-tetramethyl-rhodamine; absorbance maximum=555, emissionmaximum=580

5′FAM-3′TAMRA labelled probes are available from: Sigma-Genosys Ltd.(London Road, Pampisford, Cambridgeshire, CB2 4EF, UK. Tel. 01223839200)

5′VIC-3′TAMRA labelled probes are available from: Applied BiosystemsLtd. (Kelvin. Close, Birchwood Science Park North, Warrington, Cheshire,Wash3 7PB)

Other Suppliers are:

-   -   Integrated DNA Technologies (IDT) 1710 Commercial Park,        Coralville, Iowa, 52241, USA    -   Oswel Research Products Ltd.: Lab 5005, Medical and Biological        Sciences Building, University of Southampton, Boldrewood,        Bassett Crescent East, Southampton, SO16 7PX, Tel: 02380 592984        Equipment    -   Sterilin 20 ml plastic universal tubes    -   Clean scissors & forceps    -   Water bath set to 50° C.    -   Micro centrifuge    -   1.5 ml snap cap Eppendorf tubes (autoclaved)    -   1.5 ml screw-cap Eppendorf tubes (autoclaved) 0.5 ml snap-cap        Eppendorf tubes (autoclaved)    -   Vacuum/freeze-drier (not essential)    -   Spectrophotometer        -   Dedicated PCR cabinet, pipettes and autoclaved tips    -   Thermo-fast 96-well PCR plate and caps (Perkin Elmer/Applied        Biosystems)    -   ABI Prism 7700 Sequence Detector (Perkin Elmer/Applied        Biosystems)        2. Collecting the Feathers:    -   Pluck 8-10 ‘pin’ feathers (short, newly growing feathers with        plenty of pulp) from the brachial feather tract of each chicken        (see figures).    -   Place feathers in a plastic ‘universal’ tube for transport back        to the laboratory.        3. DNA Preparation from Feather Tips:    -   Cut off and save the proximal 1 cm of the feather (i.e. the        non-barbed part which is attached to the skin and which contains        the pulp—see photographs). Discard the distal barbed part of the        feather.    -   For each chicken, place the 8-10 saved feather ends in a 1.5 ml        snap-cap Eppendorf tube.    -   Add 500 μl proteinase K sample buffer (TNE buffer containing        0.5% SDS) containing 100 μg proteinase K (add proteinase K just        before use).    -   Incubate at 50° C. in a water-bath for 1.5-2 hours.    -   Microcentrifuge the tubes (6000 rpm, 10 min), to ‘pellet’        feather tips & debris.    -   Transfer supernatant to a new snap-cap tube (if the feathers        were from brown birds, the supernatants will be brown due to the        presence of melanin).    -   Add an equal volume (500 μl) of phenol to the supernatant again.        -   Vortex        -   Centrifuge at 13000 rpm, 2 min.        -   Transfer the upper (aqueous) phase to a new snap-cap tube.    -   Add an equal volume (500 μl) of phenol to the supernatant again.    -   Vortex    -   Centrifuge at 13000 rpm, 2 min.    -   Transfer the upper (aqueous) phase to a new snap-cap tube.    -   Add an equal volume (500 μl) of cold chloroform.    -   Vortex.    -   Centrifuge at 13000 rpm, 2 min.    -   Transfer the upper phase to a 1.5 ml screw-cap tube.    -   Add 1 ml filtered 100% ethanol.    -   Add 50 μl of 3M Sodium Acetate.    -   Gently mix by inverting the tube, and leave at room temperature        for 20 minutes (the DNA will become visible as it precipitates).    -   Centrifuge 13000 rpm, 2 minutes, to pellet the DNA (if white        chickens were used, pellet will be white; if brown chickens        used, pellet brown).    -   Discard the supernatant.    -   Rinse the pellet twice with 500 μl of 70% cold ethanol, by        gently running the ethanol down the side of the tube, then        pouring off (take care not to dislodge the DNA pellet).    -   Cover the open top of the tube with Parafilm, and make several        puncture holes in the Parafilm using a needle.    -   Place tubes in a vacuum drier for about 5 minutes to dry the        pellet (alternatively air-dry).

Re-suspend the pellet in 50 μl PCR quality water by gently vortexing.

Determine the concentration of the DNA preparation using aspectrophotometer.

Adjust the concentration to 1 mg/ml in water.

Store at −20° C.

4. TaqMan™ Quantitative PCR Assay (Perkin Elmer Biosystems)

-   -   Set up reactions in a PCR-dedicated cabinet, using PCR-dedicated        pipettes and autoclaved tips    -   Work on ice    -   Prepare master mix containing the following reagents for the        appropriate number of samples—set up duplicate reactions for        each sample (volumes given per reaction):

Volume per 25 μl Component reaction Final Concentration Taqman buffer2.5 μl MgCl₂ (5 mM) 5.0 μl 1.0 mM dATP (10 mM) 0.5 μl 0.2 mM dCTP (10mM) 0.5 μl 0.2 mM dGTP (10 mM) 0.5 μl 0.2 mM dUTP (10 mM) 0.5 μl 0.2 mMWater containing 11.6 μl ~10 μg BSA/reaction 800 μg/ml BSA Meq probe (10μM) 0.5 μl 0.2 μM Meq forward primer 1.0 μl 0.4 μM (10 μM) Meq reverseprimer 1.0 μl 0.4 μM ((10 μM) Taq Gold DNA pol 0.13 μl 26 U/ml (5 U/μl)Uracil N-glycosylase 0.25 10 U/ml (1 U/μl)

-   -   Vortex to ensure complete mixing    -   Place a thermo-fast PCR plate into a plate holder on ice and add        24 μl master mix to each well to be used    -   Add 1 μl autoclaved water to no-template-control (NTC) wells and        cap these wells prior to opening any DNA samples    -   Add 1 μl positive control DNA (=1 μl of 1 mg/ml preparation),        e.g. DNA from Rispens-infected CEF to appropriate wells and cap        these wells        -   Add 1 μg sample DNA (=1 μl of 1 mg/ml preparation), to            appropriate wells and cap        -   Briefly pulse plate in centrifuge    -   Place plate in ABI Prism 7700 Sequence Detector (Applied        Biosystems) and set up computer to read FAM fluorescence (Meq        probe), run samples    -   Thermocycling parameters

50° C. 2 min 95° C. 10 min 94° C. 15 sec {close oversize brace} × 40 60°C. 1 min

-   -   Analyse data using Microsoft excel—for each sample there will be        a Ct value (the PCR cycle at which the amount of fluorescent        product is first detected above baseline level); calculate mean        Ct value for duplicates for each DNA sample.        Results of the Above Experiment are Shown in FIG. 6.        Notes

The Forward primer (GGT CTG GTG GTT TCC AGG TGA—SEQ ID No. 2) is locatedat NT positions 1341-1361 in the GA strain Meq gene sequence. Thereverse primer (GCA TAG ACG ATG TGC TGC TGA—SEQ ID No. 3) is located atNT positions 1413-1393. The probe was designed to specifically annealbetween the two primers on the Meq target sequence (see FIG. 8).

During PCR, the fluorogenic probe binds between the two primers and,during each extension cycle, the 5′ nuclease activity of the Taqpolymerase cleaves the probe, releasing the reporter fluorochrome FAMthat is then able to fluoresce. Therefore, with each cycle, fluorescenceintensity increases. The Ct value (the cycle at which fluorescencepasses a fixed threshold) is a measure of the starting copy number ofthe target sequence: the higher the starting copy number, the lower theCt value.

Use of Bovine Serum Albumin during PCR: feather tissues (especiallythose from brown chickens) contain melanin, which has been shown to beinhibitory to PCR. Use of BSA in the reaction overcomes thismelanin-induced inhibition. Experiments were carried out in brownchickens. The addition of BSA followed the method of Giambernardi et al(1998). Biotechniques 25: 564-6.

EXAMPLE 2 Method for Detecting Specific MDV-1 (CV1 988 Vaccine) Strain

This example of the invention relates to the use of a specially modifiedversion of the 132 base pair (bp) repeat polymerase chain reaction (PCR)test to detect the presence of CVI 988 Marek's vaccine in chickenfeathers.

The 132 base pair repeat genetic sequence is located in the internalrepeat long (IR₁) segment of the Marek's disease virus (serotype 1)genome. The complete genomic sequence of MDV 1 is described in Tulman etal. (September 2000) J Virol. Vol. 74 No. 17, p 7980-7988 and has beendeposited in GenBank under accession no. AF 243438. CV1 988 isolates(vaccine strains) of serotype 1 Marek's disease have multiple copies ofthis repeat segment, whilst field strains have single copies (Silva etal (1992) Avian Dis 36: 521-528; and Becker et al (1993) Virus Genes 7:277-287). Measuring the number of copies affords the possibility ofdifferentiating vaccine strains from field strains. (Becker et al (1992)J Virol Methods 40: 307-322 and Kopacek et al (1993) Acta Virol 37:191-195).

Feathers are sampled according to the enclosed figures, the proximaltips of the axillary tract feathers being used. PCR analysis (see below)demonstrated multiple copies of the 132 bp segment in animals between 11and 28 days post vaccination. Vaccination was carried out on birds atone day of age.

An acceptable variation of the test is to use feathers in birds of anyage either taken as fresh samples, or stored for testing at a later dateto determine the presence of the CV1988 vaccine.

Protocol for 132 bp Repeat PCR on Feather Tip Samples

1. Materials Required:

Reagents:

-   -   TNE buffer (store at room temperature) contains Tris (10 mM),        NaCl (150 mM), EDTA (1 mM) and the pH is adjusted to pH 7.5        using HCl    -   Sodium Dodecyl Sulphate (SDS) 10% solution (store at room        temperature)    -   Proteinase K (lyophilised powder from Sigma # P-6556, and make        up a stock of 20 mg/ml in water, stored at −20° C.)    -   Phenol pH 7.9 obtained from Sigma (catalogue no. P-4557) (stored        at 4° C.)    -   Chloroform (stored at −20° C.)    -   3M Sodium Acetate pH5.2 (stored at room temperature)    -   Filtered neat ethanol (stored at room temperature)    -   Filtered 70% cold ethanol (stored at 4° C.)    -   PCR quality water        -   PCR quality water containing 800 μg/ml Bovine Serum Albumin            (BSA), filtered        -   Taq gold DNA polymerase (5 U/μl), Taq buffer, MgCl₂ (25 mM)            from Bio/Gene Ltd, Kimbolton, Cambridgeshire, England    -   dATP, dTTP, dCTP, dGTP (100 mM stocks) obtained from Promega        (USA); we prepare a mix containing all four of these at 10 mM        each, stored at −20° C.    -   DNA molecular size markers    -   Agarose and TBE buffer    -   Primer sequences:

MD-132 FOR; 5′ TACTTCCTATATAGATTGAGACGT-3′ (SEQ ID No. 4) MD-132 REV: 5′GAGATCCTCGTAAGGTGTAATATA-3′ (SEQ ID No. 5)Equipment:

-   -   Sterilin 20 ml plastic universal tubes    -   Clean scissors & forceps.    -   Water bath set to 50° C.    -   Micro centrifuge    -   1.5 ml snap cap Eppendorf tubes (autoclaved)    -   1.5 ml screw-cap Eppendorf tubes (autoclaved)    -   0.5 ml snap-cap Eppendorf tubes (autoclaved)    -   Vacuum/freeze-drier (not essential)    -   Spectrophotometer    -   Dedicated PCR cabinet, pipettes & autoclaved tips    -   Thermal cycler    -   Agarose gel apparatus        2. Collecting the Feathers:    -   Pluck 8-10 ‘pin’ feathers (short, newly growing feathers with        plenty of pulp) from the axillary feather tract of each chicken        (see figures).    -   Place feathers in a plastic ‘universal’ tube for transport back        to the laboratory.        3. DNA Preparation from Feather Tips:    -   Cut off and save the proximal 1 cm of the feather (i.e. the        non-barbed part which is attached to the skin and which contains        the pulp—see photographs). Discard the distal barbed part of the        feather.    -   For each chicken, place the 8-10 saved feather ends in a 1.5 ml        snap-cap Eppendorf tube.    -   Add 500 μl proteinase K sample buffer (TNE buffer containing        0.5% SDS) containing 100 μg proteinase K (add proteinase K just        before use).    -   Incubate at 50° C. in a water-bath for 1.5-2 hours.    -   Microcentrifuge the tubes (6000 rpm, 10 min), to ‘pellet’        feather tips and debris.    -   Transfer supernatant to a new snap-cap tube (if the feathers        were from brown birds, the supernatants will be brown due to the        presence of melanin).    -   Add an equal volume (500 μl) of phenol to the supernatant    -   Vortex    -   Centrifuge at 13000 rpm, 2 min    -   Transfer the upper (aqueous) phase to a new snap-cap tube    -   Add an equal volume (500 μl) of phenol to the supernatant again.    -   Vortex    -   Centrifuge at 13000 rpm, 2 min.    -   Transfer the upper phase to a new snap-cap tube.    -   Add an equal volume (500 μl) of cold chloroform.    -   Vortex.        -   Centrifuge at 13000 rpm, 2 min.    -   Transfer the upper phase to a 1.5 ml screw-cap tube.        -   Add 1 ml filtered 100% ethanol.    -   Add 50 μl of 3M Sodium Acetate.    -   Gently mix by inverting the tube, and leave at room temperature        for 20 minutes (the DNA will become visible as it precipitates).    -   Centrifuge at 13000 rpm, 2 minutes, to pellet the DNA (if white        chickens were used, pellet will be white; if brown chickens        used, pellet brown).    -   Discard the supernatant.    -   Rinse the pellet twice with 500 μl of 70% cold ethanol, by        gently running the ethanol down the side of the tube, then        pouring off (take care not to dislodge the DNA pellet).    -   Cover the open top of the tube with Parafilm, and make several        puncture holes in the Parafilm using a needle.    -   Place tubes in a vacuum drier for about 5 minutes to dry the        pellet (alternatively air-dry).    -   Re-suspend the pellet in 50 μl PCR quality water by gentle        vortexing.    -   Determine the concentration of the DNA preparation using a        spectrophotometer.    -   Adjust the concentration to 1 mg/ml in water.    -   Store at −20° C.        4. 132 bp Repeat PCR:    -   Set up reactions on ice, in 0.5 ml Eppendorf tubes, in a        PCR-dedicated cabinet, using PCR-dedicated pipettes and        autoclaved tips.    -   Prepare a ‘master mix’ containing the following reagents for the        appropriate number of samples (volumes given per reaction):

Volume per 20 μl Component reaction Final Concentration Forward primer(10 μM) 1.0 μl 0.5 μM Reverse primer (10 μM) 1.0 μl 0.5 μM 10 × Taqbuffer 2.0 μl MgCl₂ (25 mM) 1.6 μl 2 mM DNTP mix (10 mM) 0.5 μl 0.25 mMTaq gold DNA 0.1 μl 25 units/ml polymerase Water containing 12.8 μl 10μg BSA/reaction 800 μg/ml BSA*

-   -   Vortex to ensure complete mixing.    -   Aliquot 19 μl ‘master mix’ into autoclaved 0.5 ml snap-cap        Eppendorf tubes.    -   Add 1 μg DNA (=1 μl of 1 mg/ml preparation, or larger volume if        DNA preparation less concentrated).    -   Vortex to ensure complete mixing.    -   (Our thermal cycler has a heated lid, so we do not need to        overlay the reactions with mineral oil).    -   Run on a thermal cycler using the following cycling parameters:

95°C. 2 min 1 cycle 95°C. 1 min 50°C. 30 sec {close oversize brace} × 40cycles 72°C. 1 min 72°C. 10 min 1 cycle

-   -   Analyse reaction products on an agarose gel.

The results of the above experiment are shown in FIG. 7.

Notes

The sense primer is located 65 bp upstream of the repeat and theantisense primer is located 105 bp downstream of the repeat. Theexpected band size is therefore 302 bp for a single repeat (i.e.65+132+105), 434 bp for a double repeat (302+132) and 566 bp for atriple repeat (434+132) etc. Rispens vaccine strain produces many tandemrepeats.

EXAMPLE 3 Detection of Rispens Virus Genome in Feather Tip DNA usingPCR: Comparison of Samples from Different Feather Tracts

Methods

Feather Sampling from Rispens-vaccinated Chicks

Two-week-old Rhode Island Red chicks were inoculated with 1000 pfu FortDodge Rispens vaccine virus via the intra-peritoneal route. Age-matched,non-vaccinated chicks were housed in a separate room. All chicks werewing-banded in both wings, to permit identification of individual chicksthroughout the experiment. At 8, 13, 19 and 26 days post vaccination,five vaccinated chicks (#921, #923, #924, #925) and one non-vaccinatedchick (#946) were sampled. Approximately six pinfeathers were pluckedfrom each of the Cervical, Humeral, Spinal, Axillary and Femoral tracts.The remaining five feather tracts were not used either because thenumber of feathers was too few to allow sampling on four occasions, orbecause it was considered unethical to pluck from delicate regions ofthe skin of living chicks.

DNA was prepared from the feather tips and subjected to real-time PCR todetect the virus Meq gene, as summarised below.

DNA Preparation from Feather Tip Samples

DNA was prepared from feather tips as described above. Briefly, feathertips were incubated at 50° C. for 2 hours in 500 μl TNE-SDS buffercontaining 100 μg proteinase K. The supernatant was extracted withphenol, then with ice-cold chloroform. DNA was precipitated, usingethanol/sodium acetate, pelletted, vacuum dried, then resuspended to aconcentration of 200 μg/ml in water.

Real-time (TagMan) PCR Assay to Detect Meq Gene

25 μl duplex reactions were set up as described above, using Taq goldDNA polymerase and primers to amplify the viral Meq gene and the hostOvotransferrin (Ovo) gene (table 1) from 200 ng feather tip DNA. BSA waspresent at a concentration of 10 μg per reaction. The reaction kineticswere followed by inclusion of a FAM-fluorescent-tagged Meq probe and aVIC-fluorescent-tagged Ovo probe (table 1). There were duplicatereactions for each sample. The thermocycling parameters were: 1 cycle of50° C. (2 min), 1 cycle of 95° C. (10 min), followed by 40 cycles of 94°C. (15 sec) and 60° C. (1 min). In addition to the feather samples undertest, the reaction plate also included ten-fold dilutions of RispensBAC10 DNA (Rispens virus genome cloned into a Bacterial ArtificialChromosome), containing a calculated number of copies of the Rispensgenome.

The data were analysed using Microsoft Excel. For each reaction, the Ctvalue (the PCR cycle at which the amount of fluorescent product is firstdetected above baseline level), and thence the 40-Ct value, wasobtained. A Ct value of 40 indicates that no target sequence waspresent. Values are presented as ‘40-Ct’ so that a higher value equatesto more copies of the viral genome.

For the Rispens BAC10 samples, 40-Ct value was plotted against number ofcopies of virus genome (log scale), to produce a standard curve. Theequation for the linear portion of the plot was determined. For thefeather samples under test, the 40-Ct values were ‘normalised’ accordingto the 40-Ct value for Ovo (a ‘house-keeping gene’ present in allchicken cells), to correct for slight differences in the amount of DNAused for each sample. The mean, normalised 40-Ct value for duplicatereactions was then determined and was converted to Rispens genome copynumber using the standard curve.

TABLE 1 Primers and probes used in conventional and real-time PCR Ampli-Target Primer Primer Sequence Primer con Sequence Name (5′–3′) LocationSize MDV-1 Meq GGTCTGGTGGTTTCCA  1341– 73 bp Meq gene forward GGTGA 1361 (based on (SEQ ID No. 2) GA Meq GCATAGACGATGTGCT  1413– sequence)reverse GCTGA  1393 (SEQ ID No. 3) Meq AGACCCTGATGATCCG  1366– probeCATTGCGACT (5′  1391 FAM label, 3′ TAMRA label) (SEQ ID No. 1)Ovotransfer Ovo CACTGCCACTGGGCTC  4517– 62 bp rin gene forward TGT  4535(Ovo) (SEQ ID No. 6) Ovo GCAATGGCAATAAAC  4567– reverse CTCCAA  4587(SEQ ID No. 7) Ovo AGTCTGGAGAAGTCT  4537– probe GTGCAGCCTCCA 44564 (5′VIC label, 3′ TAMRA label) (SEQ ID No. 8)ResultsFeather Sampling and DNA Preparation

Although it was initially planned to sample each of the ten feathertracts, it was only practicable to use the Cervical, Humeral, Spinal,Axillary and Femoral tracts. The remaining five feather tracts could notbe used either because the number of feathers was too few to allowsampling on four occasions, or because it was considered unethical topluck from delicate regions of the skin of living chicks. Sufficientpinfeathers were obtained from each tract at 8, 13 and 19 days postvaccination. By 26 days (when the chicks were almost 6 weeks old) thepinfeathers were beginning to be replaced by harder, mature feathers.Sufficient DNA was obtained from all samples, with no marked differencesbetween amount of DNA obtained from the same number of feathers fromdifferent tracts. In order that the Meq and Ovo Ct values fell into thelinear range of detection for real-time PCR, it was found necessary todilute the DNAs to a concentration of 200 ng/μl and to use 1 μg of thisstock:per for real-time PCR reactions.

To enable accurate comparison of virus load at different time-points, indifferent feather tracts, and in different chicks, real-timequantitative PCR was performed.

For each reaction, the 40-Ct value was calculated. For the Rispens BAC10samples, 40-Ct value was plotted against number of copies of virusgenome (log scale), to produce a standard curve (FIG. 8). The lowerlimit of accurate detection was 50 copies thus, any samples with a 40-Ctvalue of 0 contain fewer than 50 copies of the Rispens genome per 200 ngfeather tip DNA.

The equation for the linear portion of the plot was: y=1.8102Ln(X)−6.796. For each of the feather samples under test, the mean,normalised 40-Ct values were converted to Rispens genome copy numberusing this equation (see Tables 2A, B, C and D; FIGS. 9 A, B, C and D).As expected, there was considerable variation between individual chicksat any given time-point. However, the levels of virus detected in thefive different feather tracts of a given individual were similar. Ineach chick, of the four timepoints tested, virus load was greatest at 13days, decreasing at 19 and 26 days.

TABLE 2A Rispens MDV copy number (8 days) 8 days post vaccination Chick# & feather Copies of Rispens MDV genome/200 ng feather DNA tractHumeral Spinal Femoral Cervical Axillary #921  673  537 2630  442  778#923  1504  4890 5127 5921  6101 #924 112910  61242 258345  31425  62444#925  2603  1265 2010 1883  1583 Mean 29422 16983 67028  9918 17726 SDev55664 29567 127552  14524  29904 SE 27832 14784 63776  7262 14952 mean

TABLE 2B Rispens MDV copy number (13 days) 13 days post vaccinationChick # & feather Copies of Rispens MDV genome/200 ng feather DNA tractHumeral Spinal Femoral Cervical Axillary #921 2133045 2564484 24322041224616  2511886 #923 3814060 4374639 5998652 3561896 38349161 #92424768376   876646 4241591 24073508  24711780 #925  866276 44784651557400 1856729  485447 Mean 7895439 3073559 3557462 7679187 16514568SDev 11313237  1708035 1974326 10974035  18229569 SE 5656619  854018 987163 5487018  9114785 mean

TABLE 2C Rispens MDV copy number (19 days) 19 days post vaccinationChick # & feather Copies of Rispens MDV genome/200 ng feather DNA tractHumeral Spinal Femoral Cervical Axillary #921  353997  613762 1158777  736207 439542 #923 1040917 1427067 1061134  1029830 1335442  #9243108135  891103 256243 1584386 391420 #925 2958332 9800358 1469708 3373701 1705109  Mean 1865345 3183073 986466 1681031 967878 SDev 13787684424421 517048 1181980 655758 SE  689384 2212211 258524  590990 327879mean

TABLE 2D Rispens MDV copy number (26 days) 26 days post vaccinationChick # & feather Copies of Rispens MDV genome/200 ng feather DNA tractHumeral Spinal Femoral Cervical Axillary #921 295801 193642 167109 74645  95940 #923 921896 694141 541176 976157 569305 #924 771634 615826349598 343195 104585 #925 353768 926750 511690 251692 236766 Mean 585775607590 392393 411422 251649 SDev 308455 305928 172194 392646 221359 SE154228 152964  86097 196323 110680 mean

For the four vaccinated chicks, the mean genome copy number (per 200 ngfeather DNA) was determined for each feather tract at each time-point,and plotted against time post vaccination using either a logarithmicscale (FIG. 5) or a linear scale (FIG. 10B). At 8 days, mean copy numberwas between 10⁴ and 10⁵ copies per 200 ng DNA. At 13 days, mean copynumber was between 5×10⁶ and 5×10⁷. at 19 days, between 10⁶ and 10⁷; andat 26 days, between 10⁵ and 10⁶.

The logarithmic plot (FIG. 10A) indicated similar virus replicationkinetics in each of the five feather tracts. However, differences areemphasised in linear plot (FIG. 10B). This plot shows that, at 13 days,the mean virus load for four chicks is up to four-fold greater in theaxillary tracts than in the other four tracts. At this time-point, virusloads in the humeral and cervical tracts were very closely related ateach time-point, and were greater than virus load in the femoral andspinal tracts. These differences between feather tracts were notapparent at 8, 19 or 26 days.

For the non-vaccinated control chick (#946) the majority of the samplesgave 40-Ct values of 0, as would be expected in the absence of MDV DNA.However, for some of the samples, the 40-Ct values were up to 3 (datanot shown). It is often the case in real-time PCR that ‘negative’samples give a 40-Ct value above 0, and values smaller than 4 areconsidered ‘unreliable’. Thus, although a 40-Ct value of 3 equates to˜200 copies of Rispens genome, this value is 100-fold lower than anyvalues obtained for samples from vaccinated chicks, and can beconsidered negative. As seen in FIG. 5B, the mean copy number for thefive feather tracts of the non-vaccinated chick does not rise above thebaseline.

Summary of Real-time PCR Analysis

-   -   Virus load in each of the feather tracts was sufficient to be        detectable by real-time PCR at 8, 13, 19 and 26 days post        vaccination.    -   Virus load was highest in all of the tested feather tracts at.        13 days post vaccination, as compared with 8, 19 and 26 days        post-vaccination.    -   At 13 days post vaccination, virus load was higher in the        axillary tract than in the humeral and cervical tracts (two        times greater) or in the spinal and femoral tracts (four times        greater).

1. A method of detecting a virus in an avian tissue sample comprising:extracting genetic material from an avian tissue sample; and testing theextracted genetic material to detect any genetic material from thevirus; characterised in that the avian tissue sample is derived from oneor more feathers of the axillary tract.
 2. A method of detecting a virusas claimed in claim 1 wherein the method provides quantitativeinformation on the amount of the virus in the sample.
 3. A method ofdetecting a virus as claimed in claim 1 wherein the method is specificfor Marek's Disease Virus (MDV).
 4. A method as claimed in claim 3wherein the method is specific for MDV serotype
 1. 5. A method ofdetecting MDV as claimed in claim 4 wherein the method is specific forMDV-1 Rispens strain CVI
 988. 6. A method as claimed in claim 5 whereinthe method comprises: (i) providing forward and reverse primers for anucleic acid polymerase, which primers are selected from the nucleotidesequence which flanks the 132 bp repeat nucleotide sequence of MDV; (ii)amplifying nucleic acid sequences between the primers; (iii) detectingthe number of 132 bp repeat sequences in the amplified nucleic acidsequences; and (iv) relating the number of 132 bp repeat sequences tothe identity of the viral nucleic acid and thereby identifying the typeof MDV in the tissue sample.
 7. A method as claimed in claim 1 whichcomprises (a) providing a polynucleotide sequence which is capable ofbinding specifically to a virus-specific target polynucleotide; (b)contacting the extracted genetic material with a probe whereby the probebinds specifically to its target viral polynucleotide; (c) determiningwhether the probe has bound to its target viral polynucleotide; and (d)determining whether the sample contains the virus on the basis that thepresence of the target polynucleotide indicates the presence of thevirus in the sample.
 8. A method as claimed in claim 7 wherein the step(c) of determining whether the probe has bound to a targetpolynucleotide comprises amplifying a region of the targetpolynucleotide, which region comprises the binding site of the probe. 9.A method as claimed in claim 8 wherein amplification is primed by thefollowing primers: Forward primer (GGT CTG GTG GTT TCC AGG TGA) (SEQ IDNO:2) Reverse primer (GCA TAG ACG ATG TGC TGC TGA) (SEQ ID NO:3).
 10. Amethod as claimed in claim 9 wherein the probe has the sequence 5′ AGACCC TGA TGA TCC GCA TTG CGA CT 3′ (SEQ ID NO:1).
 11. A method as claimedin claim 7 wherein the probe is labelled fluorescently and wherein thestep of determining whether the probe has bound to a targetpolynucleotide comprises determining the fluorescent emissions of theprobe.
 12. A method of detecting a virus as claimed in claim 1 whereinthe method involves the use of a PCR reaction.
 13. A method as claimedin claim 12 wherein before said PCR reaction is carried out, theextracted genetic material to be tested is treated with an agent toovercome the inhibitory effect of any feather tissue factor which may bepresent.
 14. A method of detecting a virus as claimed in claim 13wherein the agent is selected from one or more of bovine serum albumin;porcine (pig) albumin; and ovine (sheep) albumin.
 15. The method ofclaim 12, wherein said PCR reaction includes bovine serum albumin (BSA).